Interpretation of Tandem Mass Spectra Obtained ... - ACS Publications

May 4, 2009 - Department of Computer Science and Engineering, University of California, San Diego,. La Jolla, California 92093-0404, Center for Marine...
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Anal. Chem. 2009, 81, 4200–4209

Interpretation of Tandem Mass Spectra Obtained from Cyclic Nonribosomal Peptides Wei-Ting Liu,† Julio Ng,‡ Dario Meluzzi,† Nuno Bandeira,‡ Marcelino Gutierrez,§ Thomas L. Simmons,§ Andrew W. Schultz,§ Roger G. Linington,| Bradley S. Moore,§,⊥ William H. Gerwick,§,⊥ Pavel A. Pevzner,*,‡ and Pieter C. Dorrestein*,†,§,⊥ Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0636, Department of Computer Science and Engineering, University of California, San Diego, La Jolla, California 92093-0404, Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California 92093-0204, Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, California 95064, and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, California 92093-0636 Natural and non-natural cyclic peptides are a crucial component in drug discovery programs because of their considerable pharmaceutical properties. Cyclosporin, microcystins, and nodularins are all notable pharmacologically important cyclic peptides. Because these biologically active peptides are often biosynthesized nonribosomally, they often contain nonstandard amino acids, thus increasing the complexity of the resulting tandem mass spectrometry data. In addition, because of the cyclic nature, the fragmentation patterns of many of these peptides showed much higher complexity when compared to related counterparts. Therefore, at the present time it is still difficult to annotate cyclic peptides MS/MS spectra. In this current work, an annotation program was developed for the annotation and characterization of tandem mass spectra obtained from cyclic peptides. This program, which we call MS-CPA is available as a web tool (http://lol.ucsd.edu/ms-cpa_v1/Input.py). Using this program, we have successfully annotated the sequence of representative cyclic peptides, such as seglitide, tyrothricin, desmethoxymajusculamide C, dudawalamide A, and cyclomarins, in a rapid manner and also were able to provide the first-pass structure evidence of a newly discovered natural product based on predicted sequence. This compound is not available in sufficient quantities for structural elucidation by other means such as NMR.1 In addition to the development of this cyclic annotation program, it was observed that some cyclic peptides fragmented in unexpected ways resulting in the scrambling of sequences. In summary, MS-CPA not only provides a platform for rapid confirmation and annotation of tandem * To whom correspondence should be addressed. Contact person regarding program development: Pavel A. Pevzner, e-mail [email protected]; fax 1-858534-7029. Contact person regarding the mass spectrometry: Pieter C. Dorrestein, e-mail [email protected]; fax 1-858-822-0041. † Department of Chemistry and Biochemistry, University of California, San Diego. ‡ Department of Computer Science and Engineering, University of California, San Diego. § Scripps Institution of Oceanography, University of California, San Diego. | University of California, Santa Cruz. ⊥ Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego.

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mass spectrometry data obtained with cyclic peptides but also enables quantitative analysis of the ion intensities. This program facilitates cyclic peptide analysis, sequencing, and also acts as a useful tool to investigate the uncommon fragmentation phenomena of cyclic peptides and aids the characterization of newly discovered cyclic peptides encountered in drug discovery programs. Ribosomally as well as nonribosomally derived cyclic peptides are an important group of compounds because of their wide range of biological, toxic, and pharmacological activities, and they often exhibit unique chemical structures.2,3 For example, the cyclic toxins microcystins and nodularins produced by cyanobacteria (blue-green algae) can wipe out entire fisheries and can cause death in humans.4,5 In addition, it is now becoming increasingly clear that these naturally occurring cyclic peptides have biological roles in quorem sensing,6,7 gliding,8,9 prevention of aerial growth,10 or cell adherence regulation11 and that they can be used as a diagnostic markers for disease.12 In addition, many cyclic peptides are used in the clinic. Well-known examples of cyclic natural (1) Schultz, A. W.; Oh, D. C.; Carney, J. R.; Williamson, R. T.; Udwary, D. W.; Jensen, P. R.; Gould, S. J.; Fenical, W.; Moore, B. S. J. Am. Chem. Soc. 2008, 130, 4507–4516. (2) Schmidt, E. W.; Nelson, J. T.; Rasko, D. A.; Sudek, S.; Eisen, J. A.; Haygood, M. G.; Ravel, J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 7315–7317. (3) Pomilio, A. B.; Battista, M. E.; Vitale, A. A. Curr. Org. Chem. 2006, 10, 2075–2121. (4) Rinehart, K. L.; Harada, K.; Namikoshi, M.; Chen, C.; Harvis, C. A. J. Am. Chem. Soc. 1988, 110, 8557–8558. (5) Gupta, N.; Pant, S. C.; Vijayaraghavan, R.; Lakshmana Rao, P. V. Toxicology 2003, 188, 285–296. (6) Holden, M. T. G.; Chhabra, S. R.; de Nys, R.; Stead, P.; Bainton, N. J.; Hill, P. J.; Maneeld, M.; Kumar, N.; Labatte, M.; England, D.; Rice, S.; Givskov, M.; Salmond, G. P. C.; Stewart, G. S. A. B.; Bycroft, B. W.; Kjelleberg, S; Williams, P. Mol. Microbiol. 1999, 33, 1254–1266. (7) Ibrahim, M.; Guillot, A.; Wessner, F.; Algaron, F.; Besset, C.; Courtin, P.; Gardan, R.; Monnetl, V. J. Bacteriol. 2007, 189, 8844–8854. (8) Poupel, O.; Tardieux, I. Microb. Infect. 1999, 1, 653–662. (9) Branda, S. S.; Chu, F.; Kearns, D. B.; Losick, R.; Kolter, R. Mol. Microbiol. 2006, 59, 1229–1238. (10) Straight, P. D.; Willey, J. M.; Kolter, R. J. Bacteriol. 2006, 188, 4918–4925. (11) Sturme, M. H. J.; Nakayama, J.; Molenaar, D.; Murakami, Y.; Kunugi, R.; Fujii, T.; Vaughan, E. E.; Kleerebezem, M.; de Vos, W. M. J. Bacteriol. 2005, 187, 5224–5235. (12) Jegorov, A.; Hajduch, M.; Sulc, M.; Havlicek, V. J. Mass Spectrom. 2006, 41, 563–576. 10.1021/ac900114t CCC: $40.75  2009 American Chemical Society Published on Web 05/04/2009

products are cyclosporine, an immunosuppressant drug used to prevent organ rejection,13 seglitide, a potent growth factor release inhibitor,14 and ramoplanin, a novel antibiotic.15 Because of the importance of their therapeutic applications, there is a continued development of strategies to generate cyclic libraries for drug screening programs.16-20 In fact, many cyclic natural products with potent therapeutic properties are discovered every week.1,21-24 Therefore it is important to continue developing methods not only for isolating or preparing such cyclic peptides but also to characterize such peptides. Despite a lot of effort by mass spectrometrists,25-37 we are still exploring the way cyclic peptides behave in a mass spectrometer, in particular during collision-induced dissociation (CID). Bioinformatics tools such as MASCOT, SEQUEST, and InsPecT are capable of robust interpretation of tandem MS spectra and also enable protein identification with the equipped database search engines.38-40 However, few tools are designed for cyclic peptides with a userfriendly interface at a level accessible to non-mass spectrometrists. (13) Italia, J. L.; Bhardwaj, V.; Ravi Kumar, M. N. V. Drug Discovery Today 2006, 11, 846–854. (14) Hannon, J. P.; Nunn, C.; Stolz, B.; Bruns, C.; Weckbecker, G.; Lewis, I.; Troxler, T.; Hurth, K.; Hoyer, D. J. Mol. Neurosci. 2002, 18, 15–27. (15) Gerding, D. N.; Muto, C. A.; Owens, R. C., Jr. Clin. Infect. Dis. 2008, 46, S43–S49. (16) Kofoed, J.; Reymond, J. L. J. Comb. Chem. 2007, 9, 1046–1052. (17) Fluxa, V. S.; Reymond, J. L. Bioorg. Med. Chem. 2009, 17, 1018–1025. (18) Berkovich-Berger, D.; Lemcoff, N. G. Chem. Commun. 2008, 14, 1686– 1688. (19) Liu, T.; Joo, S. H.; Voorhees, J. L.; Brooks, C. L.; Pei, D. Bioorg. Med. Chem. 2008, [Epub ahead of print]. (20) Zhang, Y.; Zhou, S.; Wavreille, A. S.; DeWille, J.; Pei, D. J. Comb. Chem. 2009, 17, 1026–1033. (21) Feng, Y.; Carroll, A. R.; Pass, D. M.; Archbold, J. K.; Avery, V. M.; Quinn, R. J. J. Nat. Prod. 2008, 71, 8–11. (22) Linington, R. G.; Edwards, D. J.; Shuman, C. F.; McPhail, K. L.; Matainaho, T.; Gerwick, W. H. J. Nat. Prod. 2008, 71, 22–27. (23) Shimokawa, K.; Mashima, I.; Asai, A.; Ohno, T.; Yamada, K.; Kita, M.; Uemura, D. Chem. Asian J. 2008, 3, 438–446. (24) Shindoh, N.; Mori, M.; Terada, Y.; Oda, K.; Amino, N.; Kita, A.; Taniguchi, M.; Sohda, K. Y.; Nagai, K.; Sowa, Y.; Masuoka, Y.; Orita, M.; Sasamata, M.; Matsushime, H.; Furuichi, K.; Sakai, T. Int. J. Oncol. 2008, 32, 545– 555. (25) Krishnamurthy, T.; Szafraniec, L.; Hunt, D. F.; Shabanowitz, J.; Yates, J. R.; Hauert, C. R.; Carmichael, W. W.; Skulberg, O.; Coddii, G. A.; Missler, S. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 770–774. (26) Ngoka, L. C. M.; Gross, M. L. J. Am. Soc. Mass Spectrom. 1999, 10, 732– 746. (27) Jegorov, A.; Paizs, B.; Zabka, M.; Kuzma, M.; Havlıcek, V.; Giannakopulos, A. E.; Derrick, P. J. Eur. J. Mass Spectrom. 2003, 9, 105–116. (28) Yagu ¨ e, J.; Paradela, A.; Ramos, M.; Ogueta, S.; Marina, A.; Barahona, F.; Lo´pez de Castro, J. A.; Va´zquez, J. Anal. Chem. 2003, 75, 1524–1535. (29) Jegorov, A.; Paizs, B.; Kuzma, M.; Zabka, M.; Landa, Z.; Sulc, M.; Barrow, M. P.; Havlicek, V. J. Mass Spectrom. 2004, 39, 949–960. (30) Harrison, A. G.; Young, A. B.; Bleiholder, C.; Suhai, S.; Paizs, B. J. Am. Chem. Soc. 2006, 128, 10364–10365. (31) Jia, C.; Qi, W.; He, Z. J. Am. Soc. Mass Spectrom. 2007, 18, 663–678. (32) Qi, W.; Jia, C.; He, Z.; Qiao, B. Acta Chim. Sin. 2007, 65, 233–238. (33) Tilvi, S.; Naik, C. G. J. Mass Spectrom. 2007, 42, 70–80. (34) Bleiholder, C.; Osburn, S.; Williams, T. D.; Suhai, S.; Van Stipdonk, M.; Harrison, A. G.; Paizs, B. J. Am. Chem. Soc. 2008, 130, 17774–89. (35) Harrison, A. G. J. Am. Soc. Mass Spectrom. 2008, 19, 1776–1780. (36) Riba-Garcia, I.; Giles, K.; Bateman, R. H.; Gaskella, S. J. J. Am. Soc. Mass Spectrom. 2008, 19, 1781–1787. (37) Riba-Garcia, I.; Giles, K.; Bateman, R. H.; Gaskell, S. J. J. Am. Soc. Mass Spectrom. 2008, 19, 609–613. (38) Eng, J. K.; McCormack, A. L.; Yates, J. R., III J. Am. Soc. Mass Spectrom. 1994, 5, 976. (39) Perkins, D. N.; Pappin, D. J. C.; Creasy, D. M.; Cottrell, J. S. Electrophoresis 1999, 20, 3551–3567. (40) Tanner, S.; Shu, H.; Frank, A.; Wang, L. C.; Zandi, E.; Mumby, M.; Pevzner, P. A.; Bafna, V. Anal. Chem. 2005, 77, 4626–4639.

In addition most of the bioinformatics tools are based on somewhat refined fragmentation models, i.e., they may only annotate b and y ions. Both of these are the likely reasons why most scientists that isolate cyclic natural products and that develop cyclic peptide libraries for drug screening programs ignore all but only annotate a small amount of the ions that are typically observed from cyclic peptides in their structural elucidation efforts, leaving tens to hundreds of ions unaccounted for.30 We became interested in this problem because when we attempted to annotate the tandem mass spectra of cyclic natural products isolated from marine organisms by manual means, we observed that a large proportion of the spectral intensity remained unaccounted for and that the annotation was very time-consuming. Although a program that predicts theoretical fragmentation patterns such as PFIA may assist in manual annotation of cyclic peptides by providing all possible b ions,41 MS-CPA is capable of direct annotation of the actual input cyclic peptide MS spectra and is also the first program that take into account the fragments that are a result of sequence-scrambling fragmentation pathways. To improve our understanding of the fragmentation behavior of cyclic peptides we have developed a program that readily annotates a mass spectrum resulting from the collision-induced dissociation of cyclic peptides. In addition, we have created a user-friendly web interface so that other scientist that are noncomputer experts can easily use it to annotate their tandem mass spectra of cyclic peptides. Using this program, we observed that much of the spectral intensity of a MS2 mass spectra of a cyclic peptide could not be explained. Upon further analysis, we realized that unanticipated fragmentation pathways were involved in cyclic peptides when the standard fragmentation rules were applied. The data suggested those unanticipated fragments resulted in scrambling of the sequence. These unusual fragments were first described by Harrisons et al., as nondirect sequence (NDS) ions based on the scrambling of the original peptide sequence in contrast to the direct sequence (DS) ions derived from typical fragmentation pathways.30 While initially surprising to the authors that NDS are observed, the mechanistic details toward the formation of NDS ions have recently been described in detail.34 We have included NDS in our annotations. Therefore, our program, MS-CPA, not only provides evidence for the existence of these NDS ions but also enables quantitative analysis of the spectral abundance that match to DS and NDS ions. In order to demonstrate the utility of this program, we have not only applied it to the representative testing peptides, seglitide and the tyrocidines, but also used it to confirm the sequence of two newly discovered natural products, desmethoxymajusculamide C (DMMC) and dudawalamide A, both isolated from marine cyanobacteria Lyngbya majuscule (Figure 1). In addition, the program was used to verify the structure of desprenylcyclomarin C, a natural product isolated from a prenyltransferase mutant of the marine bacteria Salinispora arenicola CNS-205. This marine natural product could not be isolated in sufficient quantities to confirm its structure by NMR; therefore, this program was critical in the confirmation of its structure. Finally, during these studies we discovered three additional dehydrated cyclomarin analogues and used our program to localize the site of dehydration. (41) Jagannath, S.; Sabareesh, V. Rapid Commun. Mass Spectrom. 2007, 21, 3033–3038.

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high-resolution, full MS/MS scans were acquired in centroid or profile mode and averaged using QualBrowser software (Thermo). The Thermo-Finnigan RAW files containing the average spectra were then converted to mzXML file format using the program ReAdW (tools.proteomecenter.org).

Figure 1. Structures of cyclic peptides discussed in this paper.

EXPERIMENTAL SECTION Sample Preparation. Seglitide was purchased from Aldrich and was dissolved to a concentration of 20 µg/mL in 50:50 methanol (MeOH)/water with 1.0% acetic acid (AcOH). Dudawalamide A and DMMC were isolated from cyanobacteria and prepared in a solution of 50 µg/mL concentration in 50:50 MeOH/ water with 1.0% AcOH and was infused into the mass spectrometer. Cyclomarins were isolated from a marine actinomycete and desalted with C18 ZipTip pipet tips (Millipore) following the manufacturer’s protocol to a final concentration of 50 µg/mL. Mass Spectrometry. All samples were subjected to electrospray ionization on a Biversa Nanomate (Advion Biosystems, Ithaca, NY) nanospray source (pressure, 0.3 psi; spray voltage, 1.4-1.8 kV). Seglitide, tyrothricin, and DMMC were analyzed a Finnigan LTQ-FTICR MS instrument (Thermo-Electron Corporation, San Jose, CA) running Tune Plus software version 1.0 and Xcalibur software version 1.4 SR1. Dudawalamide A was analyzed on a Thermo LTQ-Orbitrap-MS instrument (Thermo) running Tune Plus and Xcalibur software version 2.0. Activation time and q experiments, low-resolution spectra of seglitide, tyrothricin, and cyclomarins were acquired on a Finnigan LTQ-MS (ThermoElectron Corporation, San Jose, CA) running Tune Plus software version 1.0. The final spectrum was obtained by averaging MS2 scans with QualBrowser software version 1.4 SR1 (Thermo). Generally, the instrument was first autotuned on the m/z value of the ion to be fragmented. Then, the [M + H]+ ion of each compound was isolated in the linear ion trap and fragmented by collision induced dissociation (CID). Sets of consecutive, 4202

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RESULTS AND DISCUSSION Complexity of Cyclic Peptide Fragmentation. Because so many researchers work with cyclic peptides, the annotation of tandem mass spectra from cyclic peptides is important. The annotation, however, of tandem mass spectra of cyclic peptides is often difficult for mass spectrometrists and natural product scientists alike. The difficulty in the annotation of cyclic peptides arises from the nature of cyclic peptides itself. A cyclic peptide with n amino acid residues, theoretically, will yield n series of b ions but not any y ions.32 If there are other ions such as a ions, internal fragments, and small neutral losses such as H2O and NH3, this complexity increases significantly. Therefore, it is difficult to annotate each and every ion in the spectrum of cyclic peptides and thus becomes an informatics problem. To overcome some of the complexity in the annotation of these peptides, we have developed a program that assists in the annotation of tandem mass spectrometry data based on input amino acid values and an experimental tandem mass spectrometric data set in .dta and .mzXML formats. While we have presented, at a conference, that de novo sequencing of these nonribosomal peptides can be accomplished with near “perfect” mass spectral data sets using spectral alignments and a combination of de novo and database searching algorithms,42 it quickly became clear that when we applied our first generation de novo sequencing algorithms to “nonperfect” mass spectrometry data sets typically encountered with more complex nonribosomally encoded peptides or symmetric cyclic peptides that these algorithms often identified a slightly different sequence. To improve the de novo sequencing algorithms that can be used to confirm the structures of isolated natural products, we need to improve our understanding of the resulting ions from a tandem mass spectrometry experiment. This is, in particular, important when it comes to complex cyclic peptides. Cyclic Peptide Annotation Program. To aid in the sequencing as well as to improve our understanding of the fragmentation behavior of cyclic peptides of nonribosomal origin, we developed a program named the MS-Cyclic Peptide Annotation program (MSCPA) that readily annotates a mass spectrum resulting from the CID of a cyclic peptide. In particular, this program annotates b ions, a ions (losses of CO), and b0 ions (losses of H2O). However, y ions are not included, because cyclic peptides do not yield such ions.32 The annotation program started as a Python script to mark b, a, and b0 ions given a mass spectrum. The current implementation is capable of handling .dta and .mzXML file formats as this data format is becoming the standard format for reporting or depositing mass spectra and/or proteomic data sets43,44 as spectrum inputs. For the reason that many cyclic (42) Bandeira, N.; Ng, J.; Meluzzi, D.; Linington, R. G.; Dorrestein, P.; Pevzner, P. A. Proceedings of the Twelfth Annual International Conference in Research in Computational Molecular Biology; Springer-Verlag: Berlin, Germany, 2008; pp 181-195. (43) Lin, S. M.; Zhu, L.; Winter, A. Q.; Sasinowski, M.; Kibbe, W. A. Exp. Rev. Proteomics 2005, 2, 839–845.

peptides contain unusual or modified amino acids, we leave the freedom for users to input the amino acid masses manually. There is no size limitation to the mass of the amino acid that can be manually imported. Additionally, default standard amino acids masses are provided. Finally, the amino acid sequence is specified by the user in the order that they are encountered in the peptide. For example, seglitide has a methylation on the nitrogen of alanine. This is a nonstandard amino acid; therefore, we can input 85.05280 for methyl-alanine rather than the alanine mass 71.03711. In addition, once it was recognized that even for mass spectrometrically well behaved peptides, a large proportion of the ion intensity remained unexplained, and the capabilities of this program was expanded to consider neutral amino acid losses from the b ion ladder as well as evaluation of possible rearrangements based on the series of masses initially given. The current program has thousands of lines of code to annotate a spectrum for the generation of a graphical and tabular output on a web server. We have made the MS-CPA program publicly available as a web tool at the UCSD center for computational mass spectrometry (http://lol.ucsd.edu/ms-cpa_v1/Input.py) and have also included a tutorial in the Supporting Information. In this paper, we demonstrate the utility of MS-CPA for the characterization of the cyclic peptides shown in Figure 1. The cyclic peptides in Figure 1 are representative of the type of cyclic peptides encountered in drug screening programs. Pre-analysis Data Processing of the Tandem Mass Spectrometry Input File. While the main code for this program is thousands of lines, the main challenge in the annotation process is actually the generation of a spectrum in which most peaks can be interpreted. Because of the great variance of experimental settings, instrumentation, and fragmentation properties of the compounds, preprocessing steps of the data that is required for each compound and experiment can vary a lot. To this end, we implemented a series of filters to enhance the signal-to-noise ratio of the experimental spectrum. Our current implementation regarding preprocessing includes centroid filtering, rank filtering, water filtering, isotope filtering, peak tolerance, and symmetrization. These preprocessing steps are detailed described in the Supporting Information, but the user can also choose not to carry out any preprocessing. Given that noise peaks are unavoidable in a real mass spectrometry experiment, the main goal of the filters is to eliminate ions that are likely noise or ions that are uninformative without losing the important data. In addition, this gives the users of this program the flexibility to annotate their spectra in a manner they prefer. For example, the user may only want to annotate the top 10 ions in the spectrum. This is possible with this interface. In addition it is possible to annotate unfiltered spectra but results in a much longer computational processing time. In many cases, in natural product research, the samples are available in limited quantities or the peptide does not fragment well and therefore it is not always possible to produce the best mass spectra. The filters will allow us to work with these spectra, instead of repeating the experiment, which might not be possible (44) Pedrioli, P. G. A.; Eng, J. K.; Hubley, R.; Vogelzang, M.; Deutsch, E. W.; Raught, B.; Pratt, B.; Nilsson, E.; Angeletti, R. H.; Apweiler, R.; Cheung, K.; Costello, C. E.; Hermjakob, H.; Huang, S.; Julian, R. K.; Kapp, E.; McComb, M. E.; Oliver, S. G.; Omenn, G.; Paton, N. W.; Simpson, R.; Smith, R.; Taylor, C. F.; Zhu, W.; Aebersold, R. Nat. Biotechnol. 2004, 22, 1459– 1466.

Scheme 1. Sequences of b Ions from the Fragmentation of Seglitidea

a According to the conventional pathway for fragmentation of cyclic peptides, seglitide first undergoes random ring-opening at each amide bond, yielding six different linear peptides. Sequential C-terminal amino acid cleavage results in six series of ions, for a total 30 b ions.

in real world drug discovery applications where there is often a limited supply. Nomenclature Used in This Paper. For discussion purposes of the results in this paper, we have adapted the nomenclature forwarded by Ngoka and Gross to describe the cyclic peptides in this paper.45 The nomenclature developed by Ngoka and Gross describes the ions with a four-part descriptor with the general formula xnJZ, where “x” is the designation for the type of ion (b, a, etc.) and n is the number of amino acid residues that makes up the ion. J and Z are the one-letter codes for the two amino acid residues connecting the backbone amide bond, J-Z, which is broken to form the linear ion. J is the N-terminal amino acid residue and Z is the C-terminal amino acid residue. To illustrate the nomenclature, we use seglitide, a six-amino acid residue cyclic peptide illustrated in Scheme 1 as an example. In seglitide and tyrocidines, the one letter amino acid abbreviation was used to represent each residue, while in other compounds we assigned letters in order of their sequence using the standard alphabet since they contained too many modified residues. For example, in this paper we describe DMMC for which 6 out of 9 are modified or nonstandard amino acids, while dudawalamide A has 4 out of 7 that are nonstandard, mantillamide has 5 out of 9, and cyclomarins have 5 out of 7 (Figure 1). Because the alanine in seglitide has methylation in the nitrogen position, we use A′ to represent this methylated residue. Seglitide using this nomenclature would likely undergo random ring-openings following by the bn f bn-1 pathway45 resulting in the formation of 6 (n ) 6) different series of b ions (Scheme 1). Cyclic Peptide Annotation Program Demonstration: Seglitide. We first illustrate the application and utility of MS-CPA using a simple cyclic peptide, seglitide, a somatostatin receptor antagonist consisting of six amino acids, and described the results using the nomenclature defined above (Figure 1). Seglitide was analyzed by Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS). A singly protonated ion was observed at 808.4247 Da, which is within 3 ppm of the theoretical mass of seglitide (808.4272 Da). This ion was subjected to CID in a linear ion trap, and the product ions were again analyzed by FTICR MS (Figure 2). The resulting MS2 spectra were then analyzed by MS-CPA. The spectrum was subject to standard filtering (45) Ngoka, L. C. M.; Gross, M. L. J. Am. Soc. Mass Spectrom. 1999, 10, 360– 363.

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Figure 2. Seglitide MS and MS2spectrum. MS and MS2 spectrum were collected by ESI-LTQ-FTICR MS. (A) Broadband spectrum, (B) spectra obtained with an isolation window set for the seglitide parent ion (M + H)+, (C) MS2 spectrum of seglitide, (D) zoom in spectrum of the 600∼750 m/z region.

procedures to increase the signal-to-noise ratio. First, because the raw spectrum was collected in profile mode, only the top peak was retained in a window of ±0.05 Da. Second, the top 200 most intense peaks were retained. Lastly, isotopic and water-loss peaks were filtered out, yielding 146 final peaks. As shown in Figure 3, the output of MS-CPA includes input residues and the parent mass that is obtained as user input or directly obtained from the input .dta or .mzXML file (A), summary of input filtering parameters and resulting ions counts (B), quantitative statistics of cleavage and total explainable ion intensity (C), a spectrum with color-coded matches (b ions are showed in red; water loss are green; a ions are cyan; NDSs are blue; unannotated ions are yellow) (D), a plot of mass errors of the annotated ions (E), and a list of matched fragment ions in tabular format (E). For seglitide, the MS-CPA output indicates that 28 out of the 30 possible b ions were matched to observed masses. The explainable ion intensity of the b ions combined with possible a ions and loss-of-water ions was 71.5% of the total ion intensity. 4204

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The absolute difference between the calculated and the experimental masses was less than 0.004 Da. Among the annotations, some of the ions with high intensity contained water loss even though there was no serine or threonine in the sequence. In addition, masses corresponding to addition of 28 Da (plus CO) were observed. These ions were not expected, thus we subjected these ions to additional rounds of tandem mass spectrometry (MS3 and MS4) to verify if the annotations were real or not. With these additional rounds of fragmentation, the authenticity of MS-CPA annotations was verified and these ions are indeed correctly annotated (MSn spectra were shown in the Supporting Information). Although the mechanisms behind the formation of these unusual fragments are still elusive, MS-CPA enabled us to discover the existence of these ions. Observation of NonDirect Sequence Ions in Seglitide. Because more than 28% of the ion intensity remained unexplained, we explored the nature and significance of the remaining ion intensity. Because these data were acquired with high-resolution,

Figure 3. MS-CPA output from analysis of seglitide MS2 data. MS-CPA input parameters summary (A,B), number of cleavages, and explainable intensity. The * indicates an ion that cleaves here was annotated in the spectrum (C), annotated spectrum (D), accuracy analysis (E), annotated ions list (unsymmetrized) (to save space, only the top 30 intensity ions were displayed.) (The annotation is the recommended arrangement of the input sequence computed by the program based on specified parameters; users can further manually verify to increase the confidence.) (F) In the output spectrum and annotation list, the b ions are showed in red; H2O loss is green; a ions are cyan; NDSs are blue; and unannotated ions are yellow in the spectrum and unlisted in the table. Symmetric ions are not shown in parts D or F.

the molecular mass of each ion could be determined. First, we analyzed these for alternate combinations of amino acids that would result from peptide residues rearrangements. We found 58 such ions comprising roughly 10% of the total ion intensity. Each of these scrambled sequence ions had mass errors within 0.004 Da, in agreement with all of the other masses we had annotated. The fact that so many of the ions could be explained by a rearrangement of the amino acid sequence is unlikely be coincidental or due to noise. In fact, some of these scrambled ions are of relatively high abundance. In seglitide, the most abundant NDS ion was up to 16% of the normalized ion intensity

when the most intense ion was set to 100%. These kinds of scrambled sequence ions have previously been observed in peptides and described as nondirect sequence ions.28,30-32,34-37 Because of their relatively high abundance, they are included into our annotation program MS-CPA. By their inclusion, the accountable signal intensity increases from 71.5% to 82.1%. Notably, some ions still remain unannotated, these ions are likely a result of sidechain fragmentations, unknown fragmentations, or noise inherently present in the mass spectrometry data set. To confirm the presence of NDS ions from seglitide, the two most intense of these ions, AYWV and YKVF (b5AF-K, b5YA-W), Analytical Chemistry, Vol. 81, No. 11, June 1, 2009

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Figure 4. MS3 spectra of representative seglitide sequence ions. The presence of daughter b ions from different linearized parent ions suggests that the parent ion is cyclic (as opposed to linear, as initially assumed). MS3 spectra were collected by ESI-LTQ MS. (A-E) b5 ions, (F, G) top two NDS ions observed. Expected sequence ions of a linear peptide are shown in black. The expected ions for the cyclic peptide are showed in green combined with the black ones. The red represents NDS ions.

and each b5 ion (i.e., the parent ion minus one amino acid) were isolated and subjected to an additional round of CID. 4206

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The b5 ions were chosen for comparison and were anticipated to be linear by conventional fragmentation pathways bx f

Table 1. MS-CPA Analysisa of the Two Most Intense NDS Ions and b5 Ions of Seglitide circular linear annotation no. of annotation explained critical explained cuts intensity (%) cuts intensity (%) fragmentsb b5FV b5VK b5KW b5YA b5AF b5AF-K b5YA-W

6/8 5/8 4/8 6/8 5/8 4/6 4/6

64.75 20.46 42.39 46.15 55.19 73.16 50.47

13/20 12/20 11/20 14/20 8/20 7/12 8/12

89.24 88.82 88.36 92.54 82.01 85.38 76.81

3 4 4 6 1 2 3

a Results were analyzed by isotope removal, water removal, NH3 removal, and window filtering with width 10, top 10, unsymmetric. b Fragments that cover the linear breakpoint.

bx-1.46 Surprisingly, the MS3 spectra indicated that none of these selected ions simply followed the conventional rules for fragmentation which state that cyclic peptides sequentially lose amino acid residues from the C-terminus after the initial ringopening event (Figure 4).26 Instead, we observed a mixed series of b ions (Scheme 1) which suggest that the precursors for the MS3 experiment are still cyclic. For example, if the b5 ion FAYWK was of linear structure, only the bnFK ion series should be present in the associated MS3 spectrum (Figure 4A); however, we observed the relatively intense bnYA and b2WY ions. These additional ion fragments most likely originate from cyclic peptide precursors. To explore these NDS ions behavior, we first compared the total ion intensities explained by assuming a linear precursor with those explained by assuming a circular precursor. For example, CID on the ion b5KW (KVFAY) would yield K, KV, KVF, KVFA, Y, AY, FAY, and VFAY fragments if the b5KW ion was linear. However, if this ion was circular, we would observe 20 possible fragments. In the case of b5KW (Figure 4C), the ions annotated as AYKV, FAYK, and YKVF show high intensity and are easily explained if the precursor ion is considered to be circular. In fact, 88% of total ion intensity can be explained by assuming a circular precursor, while only 42% can be explained by assuming a linear precursor. Table 1 summarizes the analysis of the seven MS2 ions that were subjected to additional CID and annotated as either linear or circular. Among these seven MS2 ions, the only one that gave poor fragmentation is b5VK (VFAYW), with 12 cleavages out of 20. However, this ion produced a very intense peak (b4AF) that corresponds to loss of phenylalanine. This peak would not have been the most intense ion in the MS3 spectrum if the initial cyclic peptide had first undergone linearization and then eliminated the C-terminal residue (i.e., tryptophan) as predicted by conventional fragmentation rules.26,45 While all of the foregoing results strongly support the cyclic nature of the MS2 ions resulting from CID of seglitide, it is likely that a mixture of cyclic and linear forms ultimately contribute to the MS3 spectrum. Although the formation of these NDS ions have been recognized since 2003,30 the actually mechanisms behind them are still a hot research topic. Several groups argued the importance of understanding this phenomenon in the development of de novo sequence programs. Therefore, a few mechanisms have been (46) Paizs, B.; Suhai, S. Mass Spectrom. Rev. 2005, 24, 508–548.

proposed to account for NDS ions.28,30-32,34 The general consensus involves a cyclic intermediate occurring by recyclization. The presence of recyclized intermediates have been verified by RibaGarcia and co-workers using ion-mobility MS.36,37 The tendency of generating NDS ions was also studied under N-acetylation modification or various activation energy.35 Recently, just after this current manuscript was submitted, a more thorough mechanism and pathway was published by Bleiholder et al., in which a sequence-scrambling fragmentation pathway was proposed describe the mechanism of NDS ions based on experimental and energetic calculations in agreement with the cyclic NDS ions we observed.34 Therefore, our program, MS-CPA, provides solid evidence showing the existence and abundance of these NDS ions with nonribosomally derived cyclic peptides. Dependence of the Intensity of NDS Ions on Activation Time and Activation q. Because we anticipated that changing the activation time and energy would provide control of the intensities of NDS ions, we analyzed the effects of the different CID parameters on NDS ion abundance (Figure S-3 in the Supporting Information). Surprisingly, and somewhat unsatisfying, the amount of these NDS ions did not demonstrate a significantly change with increased activation time and energy (q). This phenomenon can be attributed to the fact that when the activation q increases, the m/z range of a frequency sweep decreases (Figure S-4 in the Supporting Information) because low m/z product ions start to lose stable trajectories as activation energy q is rising.47,48 This is a well documented flaw with linear ion traps. Because of the large activation q, the fragment ions and NDS ions no longer fall within the acquired scan. Therefore, the cleavage coverage and NDS abundance decrease drastically (Figure S-3B in the Supporting Information). In addition, there does not appear to be significantly added benefit from changing the activation time and activation q in the increased coverage when the spectra are merged. Unfortunately we do not have PQD (Plused-Q Dissociation) on our instrument that could partially overcome this limitation found with ion traps.49,50 Although not tested in this work, as the authors do not have such instrumentation in their laboratories, it is anticipated that Q-TOFs and triple quadrupoles do not have this limitation. Capability of MS-CPA in Analyzing an Antibiotic Mixture. In addition to seglitide, we investigated the antibiotic mixture tyrothricin, which contains more than 28 different compounds and is readily available commercially due to its clinical utility as a typical antibiotic. Some of these compounds, individually called tyrocidines, are known to be cyclic peptides.51 We used MS-CPA to analyze several ions from this mixture (Figure 5, Table S-2 in the Supporting Information). In the case of tyrocidine A, the program successfully annotated 74 b ions out of 90 possible. In contrast, only 17 b ions were identified through manual annotation of tandem mass spectra from tyrocidine A, despite this being one of the most thorough studies of cyclic peptides available to date (47) Payne, A. H.; Glish, G. L. Anal. Chem. 2001, 73, 3542–3548. (48) Racine, A. H.; Payne, A. H.; Remes, P. M.; Glish, G. L. Anal. Chem. 2006, 78, 4609–4614. (49) Schwartz, J. C.; Syka, J. E. P.; Quarmby, S. T. Proceedings of the 53rd ASMS Conference on Mass Spectrometry and Allied Topics, San Antonio, TX, June 5-9, 2005. (50) Schlabach, T.; Zhang, T.; Miller, K.; Kiyonami, R. The 2006 ABRF Conference; Long Beach, CA, Feb 11-14, 2006. (51) Eckart, K. Mass Spectrom. Rev. 1994, 13, 23–55.

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Figure 5. Tyrocidines MS and MS2 spectra. MS and MS2 spectra were collected by ESI-LTQ MS:(A) broadband spectrum showing different species of tyrocidines in tyrothricin antibiotic mixture, (B) isolation of tyrocidine A (protonated form), and (C) MS2 spectrum of tyrocidine A.

in the literature demonstrating a significant advantage of spectra using our approach.52 Using MS-CPA to Annotate Cyclic Peptides Containing Nonstandard Subunits. Seglitide and the tyrocidines have a uniform peptidic backbone with standard amino acids. However, many nonribosomal cyclic peptides are cyclized via lactone formation and include nonstandard amino acids.53 Theoretical calculations suggested that cyclic peptides favor a lactone bond as the initial ring-opening site, and also the fragmentation pathway of cyclic peptides differs when lactone bond(s) were involved.29 It is therefore important to establish how these other structural features impact the fragmentation data and the results analyzed by the MS-CPA program. Thus, we analyzed several nonribosomal cyclic peptide natural products containing lactone linkages and nonstandard amino acids by tandem MS followed by MS-CPA (Table 2 and Figures S-5-S-8 in the Supporting Information). These included three marine cyanobacterial depsipeptides: desmethoxymajusculamide C (DMMC), mantillamide, and dudawalamide A, all three of which were isolated because of their biological activity to cancer cells or malaria parasites (Figure 1).54-56 Analysis of DMMC by MS-CPA uncovered 36 of the 72 b ions expected from standard fragmentation. Including NDS ions, the proportion of explained total ion intensity increased from 71.1% (52) Pittenauer, E.; Zehl, M.; Belgacem, O.; Raptakis, E.; Mistrik, R.; Allmaier, G. J. Mass Spectrom. 2006, 41, 421–447. (53) Kopp, F.; Marahiel, M. A. Nat. Prod. Rep. 2007, 24, 735–749. (54) Simmons, T. L. Ph.D. Thesis, University of California at San Diego, San Diego, CA, 2008. (55) Gutie´rrez, M.; Gerwick, W. H. Manuscript in preparation. (56) Linington, R. G. Manuscript in preparation.

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Table 2. Summary of MS-CPA Analysis of Cyclic Peptide Natural Products Discussed in the Text explainable ion intensity name

cuts

without NDS ions (%)

with NDS ions (%)

DMMC dudawalamide A mantillamide cyclomarin A cyclomarin C desa-cyclomarin C dehyb cyclomarin A dehyb cyclomarin C dehyb desa-cyclomarin C

36/72 18/42 34/72 12/42 16/42 8/42 16/42 12/42 12/42

71.10 96.00 65.21 50.34 36.98 46.74 73.79 76.35 72.16

78.30 97.30 81.99 72.48 47.20 55.48 87.88 91.83 79.09

a des: Desprenyl. b dehy: dehydrated. c For the cyclomarins, manual annotations for ions reflecting loss of methanol were included in the calculations of explainable ion intensity.

to 78.3%. Similar results were obtained for mantillamide. These data indicate that nonstandard residues and ester linkages do not diminish the program’s ability to insightfully annotate a tandem mass spectrum. Dudawalamide A was isolated from the marine cyanobacterium Lyngbya majuscula, and its structure was determined by NMR methods. A full report on the structure and bioactivity of dudawalamide will be published elsewhere.55 A high-resolution MS2 spectrum of this compound was submitted to MS-CPA for annotation. The program was also provided with the masses of the dudawalamide subunits determined by NMR (Figure S-7 in the Supporting Information). The fragmentation behavior of dudawalamide, also a lactone, was found to be very different from

the fragmentation behavior of mantillamide and DMMC. Although 96.0% of the total ion intensity was explained by b ions with absolute mass errors smaller than 0.008 Da, only 18 of the predicted 42 b ions were identified by the program. Thus, a high proportion of total ion intensity was accounted for by a small fraction of the expected b ions. This phenomenon can be explained by the presence of labile connections between residues within dudawalamide. Such weak connections are represented in normal peptides by amides N-terminal to prolines, amides C-terminal to Asp and Glu, or amides involving tertiary amines.28 Three such linkages are present in dudawalamide: one at the N-terminus of proline and the other two at the N-termini of the N-methylated phenylalanine and the N-methylated isoleucine. Because of these three labile connections, the fragmentation of dudawalamide produced only a few ions, which were consistent with the known structure of dudawalamide but provided little sequence coverage. Lastly, we used MS-CPA to investigate the structures of cyclomarin A, cyclomarin C, and desprenylcyclomarin C. The natural products cyclomarin A and C were originally isolated, based on their strong anti-inflammatory activity, from the marine bacterium Streptomyces sp. CNB-982.57 Subsequently, desprenylcyclomarin C was isolated from a prenyltransferase mutant of Salinispora arenicola CNS-205 but could not be produced in amounts sufficient to enable structural characterization by NMR.1 We therefore subjected all three cyclomarins to mass spectrometry and acquired MS2 spectra of each analogue. The broadband mass spectra of each of these cyclomarins showed a protonated ion species and a even much more stronger species corresponding to dehydrated forms (Figure S-8A,D,G in the Supporting Information), providing evidence that these natural products are prone to water loss. The MS2 spectra of both the protonated and dehydrated forms of each cyclomarin analogue were collected and subjected to MS-CPA. Analysis by MS-CPA consistently revealed the presence of strong b5GF and b4AG ions in the MS2 spectra of all of these cyclomarin species (Table 2, Figure S-8 in the Supporting Information), thus confirming that desprenylcyclomarin C is structurally related to cyclomarin A and C. Overall, these analysis of cyclomarins identified from 8 to 16 b ions out of 34 possible b ions. The fraction of explained total ion intensity ranged from 37.0 to 50.3% when NDS ions were excluded and from 47.2 to 72.5% when NDS ions were included. On the other hand, this fraction was much higher for the dehydrated forms of cyclomarins, ranging from 72.2 to 79.1% without NDS ions and from 76.4 to 91.8% with NDS ions. In addition, we have successfully localized the dehydration site to the tryptophan-derived residue. Because cyclomarins are so prone to dehydration, it is possible that this is the form that provides its anti-inflammatory activity. The most (57) Renner, M. K.; Shen, Y. C.; Cheng, X. C.; Jensen, P. R.; Frankmoelle, W.; Kauffman, C. A.; Fenical, W.; Lobkovsky, E.; Clardy, J. J. Am. Chem. Soc. 1999, 121, 11273–11276. (58) Selim, S.; Negrel, J.; Govaerts, C.; Gianinazzi, S.; van Tuinen, D. Appl. Environ. Microbiol. 2005, 71, 6501–6507. (59) Nair, S. S.; Romanuka, J.; Billeter, M.; Skjeldal, L.; Emmett, M. R.; Nilsson, C. L.; Marshall, A. G. Biochim. Biophys. Acta 2006, 1764, 1568–1576. (60) Greve, H.; Kehraus, S.; Krick, A.; Kelter, G.; Maier, A.; Fiebig, H. H.; Wright, A. D.; Ko ¨nig, G. M. J. Nat. Prod. 2008, 71, 309–312. (61) Adams, B.; Po ¨rzgen, P.; Pittman, E.; Yoshida, W. Y.; Westenburg, H. E.; Horgen, F. D. J. Nat. Prod. 2008, 71, 750–754.

likely path leading to dehydration is the formation of an imine on the tryptophan residue, yielding a conjugated system upon loss of water (Scheme S-1 in the Supporting Information). These examples highlight the usefulness of MS-CPA to assist in the structural characterization of cyclic nonribosomally encoded natural products even when limited quantities are available. CONCLUSION Because cyclic peptides are an important class of therapeutics and toxins, we have developed a program, MS-CPA, to facilitate the structural characterization of these types of natural products. Users can easily access the program on the World Wide Web in order to annotate their tandem mass spectra of cyclic peptides. Using this program, we solidified the amino acid sequence of several recently discovered bioactive natural products, such as dimethoxymajuscalide (DMMC), mantillamide, dudawalamide A, and verified the structure of desprenycyclomarin C as well as dehydro-desprenylcyclomarin C that were isolated from a desprenyltransferase knockout S. arenicola CNS-205 strain. This analysis demonstrates the strength of this program when combined with tandem mass spectrometry, as well as a candidate structure enables the structural characterization of cyclic peptides produced in such low quantities that normally prohibit the use of other structural methods such as NMR. Using our annotation program, we observed that cyclic nonribosomal peptides fragment in unusual ways. This kind of sequence-scrambling fragmentations results in a spontaneous recyclization event. The observation of NDS ions makes the problem of de novo sequencing of cyclic peptides even more challenging than was previously anticipated. Therefore, the annotation and understanding of the fragmentation patterns will, undoubtly, facilitate and improve de novo sequencing algorithm developments. In summary, our current developed program provides a rapid annotation platform for tandem MS spectra of cyclic peptides. Also, although not designed for this, it can likely also be used to analyze the cyclization phenomenon of linear peptides. We are currently using this program to annotate peptides that have been isolated from marine organisms that have potent cancer, malarial, and antibiotic resistant bacterial inhibitory activities. The approach described in this paper should be useful to the studies of cyclic peptide virulence factors, the chemical ecology of cyclic peptides, as well as cyclic peptides in drug screening programs.1,58-61 ACKNOWLEDGMENT W.-T.L. and J.N. contributed equally to this work. This work was supported by PhRMA foundation, NIH Grant GM086283, NIH Grant NS053398, NIH Grant CA100851, FIC Grant ICBG TW006634, and California Sea Grant program (Grant 85-MNP-N). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review November 13, 2008. Accepted March 22, 2009. AC900114T

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